The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6. (C) 2003 Elsevier Inc. All rights reserved.

Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein

Epstein-Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence. To identify which of these NLS are functional, fusion proteins between green fluorescent protein and deletion constructs of EBNA-6 were expressed in HeLa cells, Each of the constructs containing at least one of the NLS was targeted to the nucleus of cells whereas a construct lacking all of the NLS was cytoplasmic. Site-directed mutation of these NLS demonstrated that only three of the NLS were functional, one at the N-terminal end (aa 72-80), one in the middle (aa 412-418) and one at the C-terminal end (aa 939-945) of the EBNA-6 protein.

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re-evaluated.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Evidence of altered prefrontal-thalamic circuitry in schizophrenia: An optimised diffusion MRI study

MRI diffusion tensor imaging (DTI), optimized for measuring the trace of the diffusion tensor, was used to investigate microstructural changes in the brains of 12 individuals with schizophrenia compared with 12 matched control subjects. To control for the effects of anatomic variation between subject groups, all participants' diffusion images were non-linearly registered to standard anatomical space. Significant statistical differences in mean diffusivity (MD) measures between the two groups were determined on a pixel-by-pixel basis, using Gaussian random field theory. We found significantly elevated MD measures within temporal, parietal and prefrontal cortical regions in the schizophrenia group (P > 0.001), especially within the medial frontal gyrus and anterior cingulate. The dorsal medial and anterior nucleus of the thalamus, including the caudate, also exhibited significantly increased MD in the schizophrenia group (P > 0.001). This study has shown for the first time that MD measures offer an alternative strategy for investigating altered prefrontal-thalamic circuitry in schizophrenia. (c) 2006 Elsevier Inc. All rights reserved.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

Peripheral withdrawal recruits distinct central nuclei in morphine-dependent rats

This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis. accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways. Crown Copyright (C) 2001 Published by Elsevier Science Ltd. All rights reserved.

Oxygen-regulated gene expression in bovine blastocysts

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Defining a role for the subthalamic nucleus within operative theoretical models of subcortical participation in language

Objective:To investigate the effects of bilateral, surgically induced functional inhibition of the subthalamic nucleus (STN) on general language, high level linguistic abilities, and semantic processing skills in a group of patients with Parkinson’s disease. Methods:Comprehensive linguistic profiles were obtained up to one month before and three months after bilateral implantation of electrodes in the STN during active deep brain stimulation (DBS) in five subjects with Parkinson’s disease (mean age, 63.2 years). Equivalent linguistic profiles were generated over a three month period for a non-surgical control cohort of 16 subjects with Parkinson’s disease (NSPD) (mean age, 64.4 years). Education and disease duration were similar in the two groups. Initial assessment and three month follow up performance profiles were compared within subjects by paired t tests. Reliability change indices (RCI), representing clinically significant alterations in performance over time, were calculated for each of the assessment scores achieved by the five STN-DBS cases and the 16 NSPD controls, relative to performance variability within a group of 16 non-neurologically impaired adults (mean age, 61.9 years). Proportions of reliable change were then compared between the STN-DBS and NSPD groups. Results:Paired comparisons within the STN-DBS group showed prolonged postoperative semantic processing reaction times for a range of word types coded for meanings and meaning relatedness. Case by case analyses of reliable change across language assessments and groups revealed differences in proportions of change over time within the STN-DBS and NSPD groups in the domains of high level linguistics and semantic processing. Specifically, when compared with the NSPD group, the STN-DBS group showed a proportionally significant (p

Hypothalamic dopamine D1 receptors are involved in the stimulation of prolactin secretion by high environmental temperature in the female sheep

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin-stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol-implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 degrees C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 mu l/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390, GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress-specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.

Temporal relationship between the auditory brainstem response and focal responses of auditory nerve root and cochlear nucleus during development in the tammar wallaby (Macropus eugenii)

Thirty-two pouch-young tammar wallabies were used to discover the generators of the auditory brainstem response (ABR) during development by the use of simultaneous ABR and focal brainstem recordings. A click response from the auditory nerve root (ANR) in the wallaby was recorded from postnatal day (PND) 101, when no central auditory station was functional, and coincided with the ABR, a simple positive wave. The response of the cochlear nucleus (CN) was detected from PND 110, when the ABR had developed 1 positive and 1 negative peak. The dominant component of the focal ANR response, the N-1 wave, coincided with the first half of the ABR P wave, and that of the focal CN response, the N-1 wave, coincided with the later two thirds. In older animals, the ANR response coincided with the ABR's N-1, wave, while the CN response coincided with the ABR's P-2, N-2 and P-3 waves, with its contribution to the ABR P-2 dominant. The protracted development of the marsupial auditory system which facilitated these correlations makes the tammar wallaby a particularly suitable model. Copyright (C) 2001 S. Karger AG, Basel.

The distribution and morphological characteristics of serotonergic cells in the brain of monotremes

The distribution and cellular morphology of serotonergic neurons in the brain of two species of monotremes are described. Three clusters of serotonergic neurons were found: a hypothalamic cluster, a cluster in the rostral brainstem and a cluster in the caudal brainstem. Those in the hypothalamus consisted of two groups, the periventricular hypothalamic organ and the infundibular recess, that were intimately associated with the ependymal wall of the third ventricle. Within the rostral brainstem cluster, three distinct divisions were found: the dorsal raphe nucleus (with four subdivisions), the median raphe nucleus and the cells of the supralemniscal region. The dorsal raphe was within and adjacent to the periaqueductal gray matter, the median raphe was associated with the midline ventral to the dorsal raphe, and the cells of the supralemniscal region were in the tegmentum lateral to the median raphe and ventral to the dorsal raphe. The caudal cluster consisted of three divisions: the raphe obscurus nucleus, the raphe pallidus nucleus and the raphe magnus nucleus. The raphe obscurus nucleus was associated with the dorsal midline at the caudal-most part of the medulla oblongata. The raphe pallidus nucleus was found at the ventral midline of the medulla around the inferior olive. Raphe magnus was associated with the midline of the medulla and was found rostral to both the raphe obscurus and raphe pallidus. The results of our study are compared in an evolutionary context with those reported for other mammals and reptiles. Copyright (C) 2002 S. Karger AG, Basel.

The distribution and morphological characteristics of cholinergic cells in the brain of monotremes as revealed by ChAT immunohistochemistry

The present study employs choline acetyltransferase (ChAT) immunohistochemistry to identify the cholinergic neuronal population in the central nervous system of the monotremes. Two of the three extant species of monotreme were studied: the platypus (Omithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus). The distribution of cholinergic cells in the brain of these two species was virtually identical. Distinct groups of cholinergic cells were observed in the striatum, basal forebrain, habenula, pontomesencephalon, cranial nerve motor nuclei, and spinal cord. In contrast to other tetrapods studied with this technique, we failed to find evidence for cholinergic cells in the hypothalamus, the parabigeminal nucleus (or nucleus isthmus), or the cerebral cortex. The lack of hypothalamic cholinergic neurons creates a hiatus in the continuous antero-posterior aggregation of cholinergic neurons seen in other tetrapods. This hiatus might be functionally related to the phenomenology of monotreme sleep and to the ontogeny of sleep in mammals, as juvenile placental mammals exhibit a similar combination of sleep elements to that found in adult monotremes. Copyright (C) 2002 S. Karger AG, Basel.